Hematopoietic stem and progenitor cells (HSPCs) reside in specific niches that control survival, proliferation, self-renewal or differentiation in the bone marrow (BM). Stem cells closely associate with spindle-shaped N-cadherin- and Angiopoietin-1-expressing osteoblasts that line the endosteal bone (Calvi, L. M., Adams, G. B., Weibrecht, K. W., Weber, J. M., Olson, D. P., Knight, M. C., Martin, R. P., Schipani, E., Divieti, P., Bringhurst, F. R., et al. (2003). Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425, 841-846; Zhang, J., Niu, C., Ye, L., Huang, H., He, X., Tong, W. G., Ross, J., Haug, J., Johnson, T., Feng, J. Q., et al. (2003). Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425, 836-841; Arai, F., Hirao, A., Ohmura, M., Sato, H., Matsuoka, S., Takubo, K., Ito, K., Koh, G. Y., and Suda, T. (2004). Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118, 149-161). In normal individuals, the continuous trafficking of HSPCs between the BM and blood compartments likely fills empty or damaged niches and contributes to the maintenance of normal hematopoiesis (Wright, D. E., Wagers, A. J., Gulati, A. P., Johnson, F. L., and Weissman, I. L. (2001). Physiological migration of hematopoietic stem and progenitor cells. Science 294, 1933-1936; Abkowitz, J. L., Robinson, A. E., Kale, S., Long, M. W., and Chen, J. (2003). Mobilization of hematopoietic stem cells during homeostasis and after cytokine exposure. Blood 102, 1249-1253). Although it has been known for many years that the egress of HSPCs can be enhanced by multiple agonists, the mechanisms that regulate this critical process are largely unknown.
The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) is widely used clinically to elicit HSPC mobilization for life-saving BM transplantation and has thus served as the prototype to gain mechanistic insight about this phenomenon (Lapidot, T., and Petit, I. (2002). Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 30, 973-981; Papayannopoulou, T. (2004). Current mechanistic scenarios in hematopoietic stem/progenitor cell mobilization. Blood 103, 1580-1585). While mice deficient in the G-CSF receptor (G-CSFR−/−) are unresponsive to G-CSF stimulation, G-CSFR−/− HSPCs can be elicited by G-CSF in chimeric mice that harbored mixtures of G-CSFR+/+ and G-CSFR−/− hematopoietic cells, suggesting the contribution of ‘trans-acting’ signals (Liu, F., Poursine-Laurent, J., and Link, D. C. (2000). Expression of the G-CSF receptor on hematopoietic progenitor cells is not required for their mobilization by G-CSF. Blood 95, 3025-3031). Subsequent studies have suggested that these trans-acting signals originated from the release of proteases including serine- and metallo-proteinases whose substrates include various molecules implicated in progenitor trafficking such as VCAM-1 (Levesque, J. P., Takamatsu, Y., Nilsson, S. K., Haylock, D. N., and Simmons, P. J. (2001). Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor. Blood 98, 1289-1297), membrane-bound Kit ligand (Heissig, B., Hattori, K., Dias, S., Friedrich, M., Ferris, B., Hackett, N. R., Crystal, R. G., Besmer, P., Lyden, D., Moore, M. A., et al. (2002). Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109, 625-637), the c-Kit receptor, stromal-derived factor-1 (SDF-1 or CXCL12) (Petit, I., Szyper-Kravitz, M., Nagler, A., Lahav, M., Peled, A., Habler, L., Ponomaryov, T., Taichman, R. S., Arenzana-Seisdedos, F., Fujii, N., et al. (2002). G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 3, 687-694; Levesque, J. P., Hendy, J., Takamatsu, Y., Simmons, P. J., and Bendall, L. J. (2003). Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide. J Clin Invest 111, 187-196) and its cognate receptor CXCR4 (Levesque, J. P., Hendy, J., Takamatsu, Y., Simmons, P. J., and Bendall, L. J. (2003). Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide. J Clin Invest 111, 187-196). Among these, the CXCL12-CXCR4 axis has emerged as a likely effector because it is the sole chemokine-receptor pair capable of attracting HSPCs (Wright, D. E., Bowman, E. P., Wagers, A. J., Butcher, E. C., and Weissman, I. L. (2002). Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. J Exp Med 195, 1145-1154) and its disruption is sufficient to induce mobilization (Broxmeyer, H. E., Orschell, C. M., Clapp, D. W., Hangoc, G., Cooper, S., Plett, P. A., Liles, W. C., Li, X., Graham-Evans, B., Campbell, T. B., et al. (2005). Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 201, 1307-1318). However, the function of these proteases has been challenged by other data indicating that G-CSF-induced mobilization was normal in mice lacking virtually all neutrophil serine protease activity, even when combined with a broad metalloproteinase inhibitor (Levesque, J. P., Liu, F., Simmons, P. J., Betsuyaku, T., Senior, R. M., Pham, C., and Link, D. C. (2004). Characterization of hematopoietic progenitor mobilization in protease-deficient mice. Blood 104, 65-72). This suggests that other proteases and/or other mechanisms are involved.
The sulfated fucose polymer fucoidan can rapidly elicit HSPC mobilization (Frenette, P. S., and Weiss, L. (2000). Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms. Blood 96, 2460-2468; Sweeney, E. A., Priestley, G. V., Nakamoto, B., Collins, R. G., Beaudet, A. L., and Papayannopoulou, T. (2000). Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence. Proc Natl Acad Sci USA 97, 6544-6549). Fucoidan is synthesized by certain seaweeds, and sulfatide, is a sulfated galactolipid synthesized by mammalian cells (Roberts, D. D., Rao, C. N., Liotta, L. A., Gralnick, H. R., and Ginsburg, V. (1986). Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids. J Biol Chem 261, 6872-6877; Skinner, M. P., Lucas, C. M., Burns, G. F., Chesterman, C. N., and Berndt, M. C. (1991). GMP-140 binding to neutrophils is inhibited by sulfated glycans. J Biol Chem 266, 5371-5374; Waddell, T. K., Fialkow, L., Chan, C. K., Kishimoto, T. K., and Downey, G. P. (1995). Signaling functions of L-selectin. Enhancement of tyrosine phosphorylation and activation of MAP kinase. J Biol Chem 270, 15403-15411; Waddell, T. K., Fialkow, L., Chan, C. K., Kishimoto, T. K., and Downey, G. P. (1995). Signaling functions of L-selectin. Enhancement of tyrosine phosphorylation and activation of MAP kinase. J Biol Chem 270, 15403-15411). The synthesis of sulfatide and its non-sulfated form galactosylceramide (GalCer) is initiated by the addition of UDP-galactose to ceramide in a reaction mediated by UDP-galactose:ceramide galactosyltransferase (Cgt), an enzyme highly expressed in oligodendrocytes and Schwann cells (Sprong, H., Kruithof, B., Leijendekker, R., Slot, J. W., van Meer, G., and van der Sluijs, P. (1998). UDP-galactose:ceramide galactosyltransferase is a class I integral membrane protein of the endoplasmic reticulum. J Biol Chem 273, 25880-25888). The products of Cgt, collectively referred to as galactocerebrosides (GCs), are a major component of the myelin sheaths that facilitate the transmission of saltatory conduction (Norton, W. T., and Cammer, W. (1984). Isolation and characterization of myelin. In Myelin, P. Morell, ed. (New York, Plenum Press), pp. 147-195). Predictably, Cgt−/− mice display defects in nerve conduction and die on postnatal days 18-30 from severe tremor and ataxia (Coetzee, T., Fujita, N., Dupree, J., Shi, R., Blight, A., Suzuki, K., and Popko, B. (1996). Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability. Cell 86, 209-219; Bosio, A., Binczek, E., and Stoffel, W. (1996). Functional breakdown of the lipid bilayer of the myelin membrane in central and peripheral nervous system by disrupted galactocerebroside synthesis. Proc Natl Acad Sci USA 93, 13280-13285).
A variety of diseases, in particular cancers and hyperproliferative disorders, require treatment with agents that are preferentially cytotoxic to dividing cells. These therapies include high doses of irradiation or chemotherapeutic agents. While these doses are necessary to kill off the cancer cells, a significant side-effect of these approaches to cancer therapy is the pathological impact of such treatments on rapidly dividing normal cells, such as hair follicles, mucosal cells and the hematopoietic cells, such as primitive bone marrow progenitor cells and stem cells. The indiscriminate destruction of hematopoietic stem cells or progenitor/precursor cells can lead to a reduction in normal mature blood cell counts, such as lymphocytes, neutrophils and platelets. Such a decrease in white blood cell count also results in a loss of immune system function in these patients. As such, this may increase a patient's risk of acquiring opportunistic infections. Neutropenia resulting from chemotherapy or irradiation therapy may occur within a few days following cytotoxic treatments. The patient, however, is vulnerable to infection for up to one month until the neutrophil counts recover to within a normal range. If the reduced leukocyte count (leukopenia) and/or a platelet count (granulocytopenia) become sufficiently serious, therapy must be interrupted to allow for recovery of the white blood cell count. Such an interruption in the patient's therapeutic regimen may result in the survival of cancer cells, an increase drug resistance in the cancer cells, and may actually result in a relapse of the cancer.
Colony stimulating factors, like G-CSF and GM-CSF, are used in such a clinical setting as adjunct therapy with chemotherapy or irradiation therapy to allow for the recovery of bone marrow cells following such harsh treatment regimens. However, these therapies generally take one to two weeks before the peripheral blood counts reach an acceptable level such that the patient's risk of developing infections is diminished. In addition, bone marrow transplantation is sometimes used in the treatment of a variety of hematological, autoimmune and malignant diseases. In addition to bone marrow transplantation, ex vivo bone marrow cells may be cultured and used to expand the population of hematopoietic progenitor cells, prior to reintroduction of such cells into a patient. These hematopoietic stem cells or precursor cells may be used for ex vivo gene therapy, whereby the cells may be transformed in vitro prior to reintroduction of the transformed cells into the patient. In gene therapy, using conventional recombinant DNA techniques, a selected nucleic acid, such as a gene, may be isolated, placed into a vector, such as a viral vector, and the vector transfected into a hematopoietic cell, to transform the cell, and the cell may in turn express the product coded for by the gene. The cell then may then be introduced into a patient (see e.g., Wilson, J. M., et al., Proc. Natl. Acad. Sci. 85: 3014-3018 (1988)). However, there have been problems with efficient hematopoietic stem cell transfection (see Miller, A. D., Blood 76: 271-278 (1990)). The use of hematopoietic stem cell transplantation therapy is limited by several factors. For example, obtaining enough stem cells for clinical use requires either a bone marrow harvest under general anesthesia or peripheral blood leukapheresis. In addition, both procedures are expensive and may also carry a risk of morbidity. Furthermore, such grafts may contain a very limited number of useful hematopoietic progenitor cells. In addition, the cells that are engrafted may offer limited protection for the patient for the initial one to three weeks after engraftment, and therefore the recipients of the graft may remain severely myelosuppressed during this time period.
There is accordingly a need for agents and methods that facilitate the mobilization of hematopoietic stem or precursor/progenitor cells to the peripheral blood. Furthermore, the development of such agents may aid in the collection of such hematopoietic stem cells or hematopoietic progenitor cells for use in ex vivo cell cultures, whereby such cells can further be used in engraftment or transplantation procedures. Accordingly, the current invention addresses these needs.
All publications, patent applications, patents and other reference material mentioned are incorporated by reference in their entirety. In addition, the materials, methods and examples are only illustrative and are not intended to be limiting. The citation of references herein shall not be construed as an admission that such is prior art to the present invention.